Method of testing milk.



Huso rome'. or encontrar, new Youn.

Mn'rno). or Tiistri'uc- 'Minn'.

Specification of Letters Patent.

Application iled December 18, 1915. Serial No. 67,5611.

To all whom t may concern.'

Be it known that I, HUGO Jonio, acitizen of the UnitedI States, and aresident of lBrooklyn, New York, have invented certain new and usefulImprovements in Methods of Testing Milk, of which the following is aspeciiication. l

l My invention consists chieiiy in excluding air from theY mixture ofmilk and leucometh- .ylene blue obtained in the reductase test at thesame time re-mixing the cream which separates during the test with therest of the .milk. It is necessary to prevent the re-oXildation of theleucomethylene blue by at mospheric oxygen, since such a re-oxidationwould unduly prolong the test. It is for this reason thatthe air isexcluded. It is necessary to re-miX the cream with the rest of the milk,since otherwise the test would again be unduly prolonged, owing to therapid reduction of the methylene blue in the cream layer, which may leadto a complete decolorizat'ion of the cream layer at a time when the restof the milk is still deep blue. It is for this reason that the cream isremixed with the rest of the milk. Other objects of the invention willbecome apparent hereinafter. A capillary tube is used for the purpose'oit' excluding the air.

This method of testing milk is carried out with ,the various appliancesshown in the accompanying drawing.

Referring to vthe several views of this drawing:

Figure l is a side elevation oi a millo testing' tube provided withglass beads, rubber stopper and capillary tube to suit the needs of thenew method. Part oit' the iront wall is removed to show the interior.

Fig. 2 is a vVertical cross section oit the upper part of said testingtube on an eli-- larged scale.

Fig. 3 is a horizontal cross section testing vtube on line 3-3 of Fig.Q, the direction of the arrows.

Fig. 4 is a .horizontal cross section of said testing tube `on line 4 4of Fig. 2, seen in) the direction of the arrows.

The solutibns of methylene blue which arc intended for. the reductasetest need not'nec essarily all contain the same brand of methylene blue.They are generally watery solutions cohtain'ing however from 1 to ercent. alcohol.

of said rllhey may be standardized, as regards strength, according toknown methods.

seen in rateatea .any as, isis.

A |specially constructed test tube, is used for making the reductasetest. The necessity o using a test tube which wouldl answer the purposeof excluding the air fromf the mixtureof milk and methylene bluesolution in the reductase test is apparent from statements which I madein the pamphlet entitled: An Emy Test for Bacteria 'in .7h/Zic moiUrea-m, Barthel@ Reel/weidse Test Improved, by Hugo Joue, 1915. As thiswork has been published there seems to be no need of my describing indetail the errors .resulting from admission ofair to the mixture. l willhowever brieiiy point out the two principal causes of those errors.

The lenen-compound formed from the methylene *blue by reduction readilytakes i up oxygen' from the air, thereby restoringy the methylene blueand prolonging the test beyond the proper duration.'

The aerobic prganisms sometimes get a good chance and sometimes a poorchalice to grow in the mixture since the amount ci oxygen absorbed bythe mixture from the air is not always the same when airis ad- .mittedUnder these circumstances it is quite evison that there is no methyleneblue left in the white layer oi cream which settles at; the top. Theduration ol'the test would therefore be unduly' prolonged.

I To accomplish both purposes, viz': to eX- .clude the air and to permitthe remixing of the cream with the rest ofthe liquid, the capillaryitubeis used.` l

The test is made as follows:

30.0 cubic centimeters of the well mixed milk and a measured volume ot'`the well preserved methylene blue solution of known strength, generally4.0 cubic centimeters, `are introduced into the testing tube which `onthe drawing is identified by number 10,4

Glass beads 15, preferably of globular form and 'about 6 millimeters indiameter are then allowed to drop intro the liquid singly or in groups,as may be necessary until the liquid rises to nearly the tcp ci thetestingtube.

The perforated rubber stopper, 12, with the capillary tube 11, having abore',|19, of about 1- millimeters, diameter,inserted in it, is theninserted loosely in the mouth of the testing tube, 10, in such a waythat the air in the testing tube, 10, or mostof it, escapes through thebore, 19, of the capillary tube, 11, while the testing tube, 10, isclosed sutilciently tight to permit the mixing,` of the contentlsWithout any loss of liquid. The mixingof the liquid is accomplished byinverting the testing,` tube, 10, in such a way that the glass beadsdrop to the stopper end of the testing tube, then inverting it again tohave them drop back, and repeating the operation until they have droppedthrough the1 liquid 6 or 8 times.

The stopper 12, is to be lifted out of the .mouth of the testing tube,10, if any of the unmixed liquid should have been forced into the bore,19, of the capillary tube, 11. rlhis is done for the purpose of havingsuch liquid drop back to the remaining liquid in the testing'tube, sincethe loss of any unmiXed liquid would make the test inaccurate.

After the liquid is thus thoroughly mixed,

. the stopper, 12, can be inserted more firmly reason that the loss of asmall amount of than it was inserted the first time for the the liquidcannow no longer do anyharm,

. sinee'it could not, under these circumstances,

a'ect the duration of the test. desirable to insert the stopper thesecond -time'with sutlicient force to eject a little of the liquidthrough the bore, 19, of the capillary tube, 11, because in this Way theremoval of the last remaining au' bubbles may be accomplished.

The stopper vis then raised slightly to have the milk mixture in thebore drop to about the middle ofthe capillary tube. -After the liquid isthus deprived of air and thoroughly Y mixed, and the height of the'liquid in the bore regulated, the test is started by putting;l the tubein a Water bath or air bath or oil bath kept at the temperature of 45centigrade or 37.50 centigrade or at some other incubating temperature,`as may be. desired.

Thevcream, 13,. which rises at the top is re-mixed With-tbe remainingliquid, 14, every time that it is found to have formed a pale to adectthe' result of the test.`

blue or colorless layer of suilicient amount continued until thedecolorization of the methylene blue is completed, and the timeinserting` the capillary tube, 11, into the hole of the perforatedrubber stopper', 12, in such a .Way that its lower end does not reachthe bottom of the rubber stopper, 12, but ret is evenl This is A mainsal few millimeters above that bottom or above the cavity, 1G, at thebottom. ln order t0 facilitate the proper insertion of the capillarytube into the rubber stopper, 12, the former may be provided with aCollar, 18, so it will slip in no farther than to the proper point.

As the reductase test may be made with more or less methylene bluesolution than l cc., Ithe size of the testing tube may be variedaccordingly. as small as 45 cubic centimeters or as large as 55 cubiccentimeters. The former size would be suitable, if only 1 cubiccentimeter of methylene blue solution is used, while the latter would besuitable if as much as 10 cubic centimeters are used. ln either case, itis understood that the amount of milk used is 30 cubic centimeters. lfless milk is taken, for instance 10 cubic centimeters and only one thirdthe amount of the methylene blue'solution which would be taken for 30cubic centimeters of milk,` the testing tube may'certain'ly be made'correspondingly smaller.

The testing tube is preferably made of highly transparent, colorlessglass so the color of the contcntscan be observed easily.

The capillary tube may be made wholly of transparent glass tubing. of 6millimeters outside diameter and '1% millimeters inside diameter. It maybe 20 centimeters long. If a more durable tube is desired, the outsidediameter may 'be reduced t'o about 4 millimeters and the tube protectedby a .frame of mctallic'nickel, thus resembling in form a miniatureprotected Water gage, suitably inserted in the rubber stopper. Theinside diameter of the capillary tube may be decreased to 1 millimeteror it may be increased to Z millimeters without mate- 'riallychangingthe mode of operation. `lf

however, theinside diameter is increased to 3 millimeters, 1t 1sdesirable to close the `open end o f the capillary tube with a finger atthe moment when the testing tube is inverted for the purpose of mixingthe contents, so as to prevent the possible introduc `tion of air intothe testing tube. When operating with the ordinary capillary tube of 113millimeters diameter, or a still smaller `diameter Athis precaution willbe found unlt may have -a capacity.

lll

- the object ofhaving ige/asas than il; millimeters. t may be increasedto 30 centimeters, more especially if the ine side diameter is less'thanmillimeters. With such a length the liquid inside is not likelytooverilow even if heated tov 459 C;

The collar, it, may be replaced by three or four small glass cones fusedon the capillary tube at the same point, since such glass cones orsimilar protuberances would prevent the capillary tube from slipping toofar into the rubber stopper just as effectively as a collar would.

The outside diameter of the capillary 'tube can certainly be variedconsiderably, av thick tube being merely that of making it durable.

The glass` beads, when dropped in one by one evidently offer theadvantage that they raise the liquid gradually, not suddenly. The4diameter of the glass beads is generally about 6 millimeters. However,it may be as small as 2 millimeters or as large as 8 milli- 'meters Theglobular beads are preferred to others, but they. may have other forms..Milk white beads are preferred to others, as-they show foreign colorsbetter.` Shot like balls of nickel or of tin may be substituted for theglass beads if desired.

The rubber stopper may be so shaped that it forms a bore like hole inthe center or a groove on the side, either one of Which would performthe functions of the capillary tube. ln this way, the insertion of 4aseparate capillary tube would be rendered unnecessary.

A rubber stopper is preferably used for closing the testing tube,chiefly becauseit forms an air tight seal easily. However it may bereplaced by a cork or by. a glass stopper, if desired, or it may beomitted al together and the capillary-tube connected directly with thetesting tube.

The testing tube and the mode of operating it is not to be usedexclusively for the ordinary reductase test, but for a number ofmodifications of that test. For instance, the fermentation of the milkmay be omitted. The reductase originally present in the milk sample maybe determined by the extent of decolorization effected within a fewminutes, and therefore without any material fermentation. The tube may lbe applied to all cases where the reductase liti is measured iby thedecolorization of the methylene blue.'

The testing tube is certainly applicable to cream, not only to milk. s

' Attention is called to the fact that the liquid in the testing tubewhen itis inverted, is prevented from dropping out not-merely by thebarometric pressure of the air, but also by capillary attraction, orsurface tension.

The liquid could certainly be prevented from dropping out of the testingtube by closing it with a glass disk and a' screw cap y la'riscopes areclosed.

y the milk jointly. The liquid in the same way as the inside tubes ofpomethodl requires considerable time and a somewhat complicatedapparatus, the method with the capillary tubeis decidedly preferred.

The hood shaped cavity at the bottom of the rubber stopper couldcertainly have a shape other thanthat of a hood or of a cone and .stillanswer the purpose of causing the air to rise into the bore of thecapillary tube. li`urthermore, by using a beveled stopper and omittingthe cavity altogether, a similar effect could be obtained.

The testing tube and the mode of operat ing it is certainly notapplicable to the determination of the reductase by methylene bluemerely, but to its determination by any v'similar coloring matter asWell. Indigo-carmine, safranin, various imido-quinones, indogenid, andother substances which are easily reduced and easily reoxidized could besubstituted for the methylene blue with more or less success.

Furthermore, the testing tube .and the mode of operating it may be usedin cases where not theJ reductase but a similar enzym is to bedetermined in milk; in fact, it can be used if an entirely dilferentingredient of is to be determined so long as a reagent is introducedinto the milk, in such a test, which is deteriorated by atmosphericoxygen.

`Likewise the tube and the mode of oper,- ating it maybe used'for anybacteriological determination involving the cultivation of anaerobicorganisms in the absence of atu mospheric oxygen.

The displacement of the air in the testing tube need lnot necessarily beeffected by a solid such as glass beads, andthe 'liquid made to displacethe air completely. However, the use of the glass beads besides theliquid for the purpose of displacing the air is preferred, since. itfacilitates the mixing of the liqui Claims:

I. The method of excluding, in the course of an 'operation such as thereductase test, air from a vessel containing milk, mixed with ano'xidizable substance, at the same time re-mixing the cream with therest of the milk, which consists in adding solid in soluble bodies untilthe vessel is nearlylilled to the top, then inserting a stopper into thealone can certainly be However, as this mouth ofthe vessehsaid stopperbeing pro time ite-mixing the cream with the rest of the milk, Whichconsists in adding solid insoluble bodies until the vessel is nearlyfilled to the top, then inserting a stopper into the mouth of thevessel,. said vstopper being provided with a eapillarypassage capable ofpermitting the escape of the residual air and also capable of permittingthe changes of the volume of the milk mixture produced by changes intemperature, and finally inverting the vessel, substantially as setforth.

3. The method of excluding, in the course of an operation suc-h as thereductase test, air from e vessel containing milk, mixed with anoxidizable substance, at the same l5 rubber-like material into the mouthoi' the 20 Vessel, said stopper being provided will: a capillarypassage, tapable of permitting; the escape of the residual ail', andl{inally invert ing the vessel, substantially as set forth.

HUGO JUNE.

. Witnesses:

t SAMUEL Lovlcn,

ISABEL LEvINsoN.

